Nitric oxide-producing hydrogel materials

ABSTRACT

Hydrogels releasing or producing NO, most preferably polymerizable biodegradable hydrogels capable of releasing physiological amounts of NO for prolonged periods of time, are applied to sites on or in a patient in need of treatment thereof for disorders such as restenosis, thrombosis, asthma, wound healing, arthritis, penile erectile dysfunction or other conditions where NO plays a significant role. The polymeric materials can be formed into films, coatings, or microparticles for application to medical devices, such as stents, vascular grafts and catheters. The polymeric materials can also be applied directly to biological tissues and can be polymerized in situ. The hydrogels are formed of macromers, which preferably include biodegradable regions, and have bound thereto groups that are released in situ to elevate or otherwise modulate NO levels at the site where treatment is needed. The macromers can form a homo or hetero-dispersion or solution, which is polymerized to form a hydrogel material, that in the latter case can be a semi-interpenetrating network or interpenetrating network. Compounds to be released can be physically entrapped, covalently or ionically bound to macromer, or actually form a part of the polymeric material. The hydrogel can be formed by ionic and/or covalent crosslinking. Other active agents, including therapeutic, prophylactic, or diagnostic agents, can also be included within the polymeric material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International PCT ApplicationNo. PCT/US01/27414, filed Sep. 4, 2001, which is a acontinuation-in-part of U.S. application Ser. No. 09/653,406, filed Sep.1, 2000, which claims the benefit of U.S. Provisional Application No.60/152,054, filed Sep. 2, 1999.

FIELD OF THE INVENTION

The present invention relates to polymerizable hydrogel materials thatproduce physiologically relevant amounts of nitric oxide (NO).

BACKGROUND OF THE INVENTION

Endothelial cells, normally present as a monolayer in the intimal layerof the arterial wall, are believed to play an important role in theregulation of smooth muscle cell (SMC) proliferation in vivo.Endothelial cells are seriously disrupted by most forms of vascularinjury, including that caused by percutaneous transluminal coronaryangioplasty and similar procedures. Approximately 35–50% of patientstreated by percutaneous transluminal coronary angioplasty experienceclinically significant renarrowing of the artery, or restenosis, withinsix months of the initial treatment. Restenosis is due, at least inpart, to migration and proliferation of smooth muscle cells in thearterial wall along with increases in secretion of matrix proteins toform an obstructive neointimal layer within the arterial wall. Similarissues limit the performance of vascular grafts. The processes thatregulate arterial wound healing following vascular injury, such as thatcaused by angioplasty, are as yet poorly understood, but are believed toinvolve a complex cascade of blood and vessel wall-derived factors.

Numerous factors that stimulate intimal thickening and restenosis havebeen identified through administration of exogenous proteins, geneticalteration of cells, or through the blockade of certain signals usingantibodies or other specific growth factor inhibitors. These smoothmuscle cell mitogens and chemoattractants derive from both the blood orthrombus formation and from the vessel wall itself. Endothelial cellsproduce a number of substances known to down-regulate smooth muscle cellproliferation, including heparin sulfate, prostacyclin (PG12), and NO.

NO is an endothelium-derived target molecule useful for the preventionof restenosis because, in addition to limiting the proliferation ofsmooth muscle cells (Garg et al., (1989) J. Clin. Invest., 83:1774–7),NO reduces platelet aggregation (de Graaf et al., (1992) Circulation,85:2284–90; Radomski et al., (1987) Br. J. Pharmacol., 92:181–7),increases endothelial cell proliferation (Ziche et al., (1993) Biochem.Biophys. Res. Comm., 192:1198–1203), and attenuates leukocyte adhesion(Lefer et al., (1993) Circulation, 88:2337–50), all of which are highlydesirable for the reduction of intimal thickening and restenosis(Loscalzo, (1996) Clin. Appl. Thromb. Hemostas., 2:7–10). Because of thecomplexity of the restenotic process, approaches that act upon multipletargets are the most likely to be successful.

The mechanisms whereby NO affects these multiple responses are not fullyunderstood as yet, but it is known that NO activates soluble guanylatecyclase by binding to its heme moiety, thereby elevating the levels ofcyclic guanosine monophosphate (cGMP), an intracellular second messengerwith multiple cellular effects (Moro et al., (1996) Proc. Natl. Acad.Sci. USA, 93:1480–5). The effects of NO can often be mimicked by theadministration of cGMP or more stable derivatives of cGMP (Garg et al.,(1989) J. Clin. Invest., 83:1774–7). In addition, NO has been found toinhibit ribonucleotide reductase, an enzyme that convertsribonucleotides into deoxy ribonucleotides, thus significantly impactingDNA synthesis (Lepoivre et al., (1991) Biochem. Biophys. Res. Comm.,179:442–8; Kwon et al., (1991) J. Exp. Med., 174:761–7), as well asseveral enzymes involved in cellular respiration (Stuehr et al., (1989)J. Exp. Med., 169:1543–55).

A number of molecules that produce NO under physiological conditions (NOdonors) have been identified and evaluated both in vitro and in vivo. NOdonor molecules exert biological effects mimicking those of NO andinclude S-nitrosothiols (Diodati et al, (1993) Thromb. Haem., 70:654–8;Lefer et al., (1993) Circulation, 88:2337–50; DeMeyer et al., (1995) J.Cardiovasc. Pharmacol., 26:272–9), organic nitrates (Ignarro et al.,(1981) J. Pharmacol. Exp. Ther., 218:739–49), and complexes of NO withnucleophiles (Diodati et al., (1993) Thromb. Haem., 70:654–8; Diodati etal., (1993) J. Cardiovasc. Pharmacol., 22:287–92; Maragos et al., (1993)Cancer Res., 53:564–8). Most of these have been low molecular weightmolecules that are administered systemically and have short half-livesunder physiologic conditions, thus exerting effects upon numerous tissuetypes with a brief period of activity. In addition, L-arginine is oftenthought of as a NO donor, as L-arginine is a substrate for NO synthase,and thus administration of L-arginine increases endogenous NO productionand elicits responses similar to those caused by NO donors in most cases(Cooke et al., (1992) J. Clin. Invest., 90:1168–72).

The development of NO-releasing polymers containing NO/nucleophilecomplexes has been reported by Smith et al., (1996) J. Med. Chem.,39:1148–56. These materials were capable of releasing NO for as long asfive weeks in vitro and were able to limit smooth muscle cellproliferation in culture and to reduce platelet adherence to vasculargraft materials in an arterio-venous shunt model. These materials showpromise for numerous clinical applications where localized NO productionwould be desired, such as anti-thrombotic coating materials forcatheters, but probably will not be useful for the direct treatment oftissues in vivo as these materials suffer from a number ofdisadvantages. These polymers may be produced as films, powders, ormicrospheres, but they cannot be formed in situ in direct contact withcells and tissues, thus making it difficult to strictly localize NOtreatment to a tissue and potentially causing issues with the retentionof the polymer at the site of application. The formulation issues willalso make local administration during laparoscopic or catheter-basedprocedures difficult or impossible. Additionally, biocompatibility ofthe base polymer is a serious issue for implantable, NO-releasingpolymers, especially those intended for long-term use, as inflammatoryand thrombotic responses may develop after the cessation of NO release.

With respect to chronic wound healing, approaches that are common todayare typically based on simple wound care regimens involving debridement,cleaning, and application of moist dressings (Thomas S, Leigh (1998).WOUND DRESSINGS. WOUNDS: BIOLOGY AND MANAGEMENT, D. Leaper and K.Harding. New York, N.Y., Oxford University Press). More advanceddressings such as topical gels containing growth factors have resultedin enhanced healing rates in some clinical studies (Wieman T, Smiell J,Su Y. Efficacy and safety of a topical gel formulation of recombinanthuman platelet-derived growth factor-BB (beclapermin) in patients withnonhealing diabetic ulcers: a phase III randomized, placebo-controlled,double-blind study. Diabetes Care 1998; 21: 822–827; Wieman T J and theBeclapermin Gel Studies Group. Clinical efficacy of Beclapermin(rhPDGF-BB) Gel. Am J Surg 1998; 176: 74S–79S; Martinez-de Jesus F R,Morales-Guzman M, Gastaneda M, Perez-Morales A, Garcia-Alsono J,Mendiola-Segura L. Randomized single-blind trial of topical ketanserinfor healing acceleration of diabetic foot ulcers. Arch Med Res 1997; 28:95–99), however on the whole these treatments are difficult to apply andare often too expensive for application to large, chronic wounds.Additionally, not all chronic wounds display growth factor deficiencies,and other mechanisms such as rapid degradation by wound proteinases maybe involved in the reduction of growth factor levels observed in manychronic wounds. Many chronic wounds are unresponsive to growth factortherapy (Greenhalgh D. The role of growth factors, in wound healing. JTrauma 1996; 41: 159–167).

With respect to proliferation of endothelial cells, it has been shownthat the presence of NO decreases endothelial cell proliferation. See,for example, Heller, R., Polack, T., Grabner, R., Till, U. (1999)“Nitric oxide inhibits proliferation of human endothelial cells via amechanism independent of cGMP”, Atherosclerosis, 144:49–57; and Sarkar,R., Webb, R. C., Stanley, J. C. (1995) “Nitric oxide inhibition ofendothelial cell mitogenesis and proliferation”, Surgery, 118:274–9.However, these studies utilized very high doses of NO-releasing drugs,which may account for the decreased endothelial cell proliferation.Additionally, previous researchers have found it difficult to seedendothelial cells onto devices: Scott-Burden, T., Tock, C. L., Schwarz,J. J., Casscells, S. W., Engler, D. A. (1996) “Genetically engineeredsmooth muscle cells as linings to improve the biocompatibility ofcardiovascular prostheses”, Circulation, 94:235–8.

It is believed by the inventors that the development of materials thatencourage the proliferation and/or migration of endothelial cells shouldenhance the growth of endogenous endothelial cells from tissuesurrounding an implant onto the implant surface. Therefore, applicantspropose that endothelialization of blood-contacting implants, such asstents, grafts, and ventricular assist devices, may significantlyimprove device performance by decreasing thrombogenicity and smoothmuscle cell proliferation.

It would be more efficient if NO releasing compounds or compoundsmodulating NO levels could be administered solely to the site in need oftreatment, and in some cases, reduce or eliminate side effects due tosystemic administration of the agents, particularly over prolonged timeperiods.

SUMMARY OF THE INVENTION

Biocompatible polymeric materials releasing or producing physiologicalamounts of nitric oxide (NO) for prolonged periods of time are describedherein. The biocompatible polymeric materials are applied to sites on orin a patient in need of treatment thereof for disorders such asrestenosis, thrombosis, asthma, wound healing, arthritis, penileerectile dysfunction or other conditions where NO plays a significantrole. The polymeric materials can be formed into films, coatings, ormicroparticles for application to medical devices, such as stents,vascular grafts and catheters. The polymeric materials can also beapplied directly to biological tissues and can be polymerized in situ.

The polymers are formed of macromers, which may include biodegradableregions, and have bound thereto groups that are released in situ toelevate or otherwise modulate NO levels at the site where treatment isneeded. The macromers can form a homo or hetero-dispersion or solution,which is polymerized to form a polymeric material, that in the lattercase can be a semi-interpenetrating network or interpenetrating network.Compounds to be released can be physically entrapped, covalently orionically bound to macromer, or actually form a part of the polymericmaterial. Hydrogels can be formed by ionic and/or covalent crosslinking.Other active agents, including therapeutic, prophylactic, or diagnosticagents, can also be included within the polymeric material.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of the synthesis of S-nitrosocysteine hydrogels(Acryloyl-PEG-Cys-NO).

FIG. 2 is a schematic of the synthesis of acryloyl-PEG-Lysine₅NO-nucleophile complex hydrogels.

FIG. 3 is a schematic of the synthesis ofacryloyl-PEG-DETA-NO-nucleophile complex hydrogels.

FIG. 4 is a graph showing the temporal release (% NO released over timein days) of NO from acryloyl-PEG-Lys₅-NO hydrogels at pH 7.4 (circles)and pH 3 (squares).

FIG. 5 is a graph showing the temporal release (% NO released over timein hours) of NO from acryloyl-PEG-DETA-NO hydrogels at pH 7.4 (circles)and pH 2 (squares).

FIG. 6 is a graph showing the temporal release (% NO released over timein hours) of NO from PEG-Cys-NO hydrogels at pH 7.4 (circles) and pH 2(squares).

FIG. 7A is a graph showing the temporal release (μmol NO released pergram of polymer over time in hours) of NO from PVA-NO-bFGF hydrogels atpH 7.4, 37° C. FIG. 7B is a graph showing the temporal release (% oftheoretical bFGF released per gram of gel over time in hours) fromPVA-NO-bFGF hydrogels at pH 7.4, 37° C.

FIGS. 8A and 8B are graphs showing that acryloyl-PEG-Lysine-NO hydrogelsinhibit the proliferation of smooth muscle cells. FIG. 8A, % of controlcell number, hydrogel formulation. FIG. 8B, % of control cell number,soluble polymer.

FIGS. 9A and 9B are graphs showing the inhibition of SMC proliferationby NO released from acryloyl-PEG-DETA-NO hydrogels (FIG. 9A) and solublepolymer (FIG. 9B), as a percentage of the control.

FIGS. 10A and 10B are graphs showing inhibition of SMC proliferation byNO released from acryloyl-PEG-Cys-NO hydrogels (FIG. 10A) and solublepolymer (FIG. 10B), as a percentage of controls.

FIG. 11 is a graph comparing the degree of inhibition of smooth musclecell growth by NO released from hydrogels: acryloyl-PEG-Lys-NO,acryloyl-PEG-DETA-NO, and acryloyl-PEG-Cys-NO, compared to controlhydrogel with NO. The percent inhibition of smooth muscle cell growth isdetermined by comparing the cell growth for each NO-releasing hydrogelto a control PEG-diacrylate hydrogel.

FIG. 12 is a graph of endothelial cell proliferation, which wasstimulated when cultured in the presence of NO-releasing PEG hydrogelswith varying NO release kinetics. PEG-diacrylate hydrogels were used asa control. The last three bars represent a statistical variance ofp≦0.01 compared to control.

FIG. 13 is a graph of endothelial cell proliferation, which wasstimulated when cultured on NO-releasing hydrogels that contained thecell adhesion peptide sequence RGDS. The last bar represents astatistical variance of p<0.02 versus either RGDS or DETA-NO hydrogelsalone.

FIG. 14 is a graph of endothelial cell proliferation, which wasstimulated when cultured on NO-releasing hydrogels that contained thecell adhesion peptide sequence REDV. The last bar represents astatistical variance of p<0.02 versus either REDV or DETA-NO hydrogelsalone.

FIG. 15 is a graph of the proliferation of HDFs cultured in the presenceof NO-releasing PVA hydrogels by cell counts. No significant changes inproliferation were observed with exposure to PVA-NO materials.

FIGS. 16A and 16B are graphs of the matrix production by fibroblastscultured in the presence of NO-releasing PVA hydrogels. 16A shows therelease of NO from PVA hydrogels increased the production of collagen byHDFs (p<0.01 versus control) while 16B shows only slightly increasedtotal matrix produced per cell (p>0.05).

FIG. 17A and 17B are graphs comparing wound area and perimeter overtime. At the time of each dressing change, pictures of the wounds weretaken to assess wound area (17A) and wound perimeter (17B) using imageanalysis software. No difference in wound area or perimeter was observedbetween control and NO hydrogel treatment groups.

FIG. 18 is a graph of the granulation tissue thickness of wounds byexamination of histological sections from wounds treated with PVA orPVA-NO hydrogels. A trend of increasing granulation tissue thicknesswith increasing NO concentration was observed.

FIG. 19 is a graph reflecting wound collagen synthesis. Histologicalsections from animals at day 29 revealed that wound collagen synthesiswas significantly increased through treatment with NO-releasinghydrogels. The second bar represents a statistical variance of p<0.001versus control.

DETAILED DESCRIPTION OF THE INVENTION

Biocompatible polymeric materials releasing or producing physiologicalamounts of nitric oxide (NO) and methods of use for the treatment ofdisorders such as restenosis, thrombosis, asthma, wound healing,arthritis, penile erectile dysfunction, or other conditions where NOplays a significant role, are provided herein.

I. Polymeric Materials for Release of NO

The polymeric materials are biocompatible and release or produce NO. Invarious preferred embodiments, the polymers are also biodegradable, formhydrogels, polymerize in situ and are tissue adherent. The polymericmaterials can also be formed into films, coatings, or microparticles forapplication to medical devices, such as stents, vascular grafts andcatheters. These properties are conferred by the selection of themacromer components as well as addition of various groups to thecomponents.

The term “polymerizable” means that the regions have the capacity toform additional covalent bonds resulting in macromer interlinking, forexample, carbon-carbon double bonds of acrylate-type molecules. Suchpolymerization is characteristically initiated by free-radical formationresulting from photon absorption of certain dyes and chemical compoundsto ultimately produce free-radicals, although polymerization can beobtained using other methods and reagents known to those skilled in theart.

All chemicals mentioned herein are obtainable from commercial chemicalcompanies such as Sigma-Aldrich Chemical Corp. (St. Louis, Mo.), unlessotherwise specified.

A. Polymeric Materials

The polymeric materials described herein must be biocompatible, i.e.,not eliciting a significant or unacceptable toxic or immunogenicresponse following administration to or implantation into an individual.

A number of polymeric materials are known which are biocompatible,including both natural and synthetic polymers. Examples include proteins(of the same origin as the recipient), polysaccharides such aschondroitin sulfate and hyaluronic acid, polyurethanes, polyesters,polyamides, and acrylates. Polymers can be degradable or non-degradable.

The preferred polymeric materials will be selected based on acombination of properties conferred by the various components, which mayinclude at least one water soluble region, such as polyethylene glycol(PEG) or polyvinyl alcohol (PVA), at least one biodegradable region suchas regions that degrade hydrolytically, and at least one group that canbe used to polymerize the macromers in situ.

One advantage to using the hydrogels described herein is the ability tocovalently attach a variety of bioactive molecules. As demonstrated bythe stimulation of proliferation of endothelial cells cultured onhydrogels containing both a cell adhesion peptide sequence and an NOdonor (FIG. 13), many factors can be combined within the same hydrogelin order to design a material that will perform optimally for thedesired application. Examples of cell adhesion peptide sequences (alsoreferred to herein as cell adhesion ligands) include RGD, RGDS, REDV(the letters indicate the single letter amino acid nomenclature known tothose skilled in the art), and other sequences that are endothelialcell-specific. The cell adhesion ligands are used to specifically targetthe adhesion, proliferation, and migration of certain cells. The celladhesion ligand may be a peptide, protein, carbohydrate, or other typeof moiety that will assist in seeding cells onto devices. For example,other bioactive molecules such as growth factors have also been shown toretain their efficacy when covalently attached to PEG (Mann B, SchmedlenR, West J. Tethered-TGF-beta increases extracellular matrix productionof vascular smooth muscle cells. Biomaterials 2001; 22: 439–444). Thus,there exists the possibility of creating a multifunctional material thatcombines NO therapy with at least one cell-specific adhesion ligand orgrowth factor in order to achieve specific results.

Water-Soluble and/or Tissue Adhesive Regions

A variety of water soluble materials can be incorporated into thepolymers. The term “at least substantially water soluble” is indicativethat the solubility should be at least about 5 g/100 ml of aqueoussolution. In preferred embodiments, the core water soluble region canconsist of poly(ethylene glycol), referred to herein as “PEG”,poly(ethylene oxide), poly(vinyl acetate), poly(vinyl alcohol), referredto herein as “PVA”, poly(vinylpyrrolidone), poly(ethyloxazoline),poly(ethylene oxide)-co-poly(propyleneoxide) block copolymers,polysaccharides or carbohydrates such as hyaluronic acid, dextran,heparin sulfate, chondroitin sulfate, heparin, or alginate, or proteinssuch as gelatin, collagen, albumin, or ovalbumin.

Hydrophilic (i.e., water soluble) regions will generally be tissueadhesive. Both hydrophobic and hydrophilic polymers which include alarge number of exposed carboxylic groups are tissue adhesive orbioadhesive. Ligands such as RGD peptides and lectins which bind tocarbohydrate molecules on cells can also be bound to the polymer toincrease tissue adhesiveness.

Degradable Regions

Polyesters (Holland et al., 1986 Controlled Release, 4:155–180) ofα-hydroxy acids (viz., lactic acid, glycolic acid), are the most widelyused biodegradable materials for applications ranging from closuredevices (sutures and staples) to drug delivery systems (U.S. Pat. No.4,741,337 to Smith et al.; Spilizewski et al., 1985 J. Control. Rel.2:197–203). In addition to the poly(hydroxy acids), several otherpolymers are known to biodegrade, including polyanhydrides andpolyorthoesters, which take advantage of labile backbone linkages, asreported by Domb et al., 1989 Macromolecules, 22:3200; Heller et al.,1990 Biodegradable Polymers as Drug Delivery Systems, Chasin, M. andLanger, R., Eds., Dekker, New York, 121–161. Polyaminoacids have alsobeen synthesized since it is desirable to have polymers that degradeinto naturally occurring materials for in vivo use.

The time required for a polymer to degrade can be tailored by selectingappropriate monomers. Differences in crystallinity also alterdegradation rates. Due to the relatively hydrophobic nature of thesepolymers, actual mass loss only begins when the oligomeric fragments aresmall enough to be water soluble. Hence, initial polymer molecularweight influences the degradation rate.

The biodegradable region is preferably hydrolyzable under in vivoconditions. Hydrolyzable groups may be polymers and oligomers ofglycolide, lactide, ε-caprolactone, other (α-hydroxy acids, and otherbiologically degradable polymers that yield materials that are non-toxicor present as normal metabolites in the body. Preferred poly((α-hydroxyacid)s are poly(glycolic acid), poly(DL-lactic acid) and poly(L-lacticacid). Other useful materials include poly(amino acids),poly(anhydrides), poly(orthoesters), and poly(phosphoesters).Polylactones such as poly(ε-caprolactone), poly(ε-caprolactone),poly(δ-valerolactone) and poly(gamma-butyrolactone), for example, arealso useful.

Biodegradable regions can also be constructed from polymers or monomersusing linkages susceptible to biodegradation by enzymes, such as ester,peptide, anhydride, orthoester, and phosphoester bonds. Degradablematerials of biological origin are well known, for example, crosslinkedgelatin. Hyaluronic acid has been crosslinked and used as a degradableswelling polymer for biomedical applications (U.S. Pat. No. 4,987,744 todella Valle et al., U.S. Pat. No. 4,957,744 to Della Valle et al. (1991)Polym. Mater. Sci. Eng., 62:731–735).

Biodegradable Hydrogels

A number of polymers have been described which include both watersoluble regions and biodegradable regions. Sawhney et al., (1990) J.Biomed. Mater. Res. 24:1397–1411, copolymerized lactide, glycolide andε-caprolactone with PEG to increase its hydrophilicity and degradationrate. U.S. Pat. No. 4,716,203 to Casey et al. (1987) synthesized aPGA-PEG-PGA block copolymer, with PEG content ranging from 5–25% bymass. U.S. Pat. No. 4,716,203 to Casey et al. (1987) also reportssynthesis of PGA-PEG diblock copolymers, again with PEG ranging from5–25%. U.S. Pat. No. 4,526,938 to Churchill et al. (1985) describednoncrosslinked materials with MW in excess of 5,000, based on similarcompositions with PEG; although these materials are not water soluble.Cohn et al. (1988) J. Biomed. Mater. Res. 22:993–1009 described PLA-PEGcopolymers that swell in water up to 60%; these polymers also are notsoluble in water, and are not crosslinked. The features that are commonto these materials are that they use both water-soluble polymers anddegradable polymers, and that they are insoluble in water, collectivelyswelling up to about 60%.

U.S. Pat. No. 5,410,016 issued on Apr. 25, 1995 to Hubbell, et al.,describes materials which are based on polyethylene glycol (PEG),because of its high biocompatible and thromboresistant nature, withshort polylactide extensions to impart biodegradation and acrylatetermini to allow rapid photopolymerization without observable heatproduction. These materials are readily modified to produce hydrogelswhich release or produce NO.

The polymerizable regions are separated by at least one degradableregion to facilitate uniform degradation in vivo. There are severalvariations of these polymers. For example, the polymerizable regions canbe attached directly to degradable extensions or indirectly via watersoluble nondegradable sections so long as the polymerizable regions areseparated by a degradable section. For example, if the macromercomposition contains a simple water soluble region coupled to adegradable region, one polymerizable region may be attached to the watersoluble region and the other attached to the degradable extension orregion. In another embodiment, the water soluble region forms thecentral core of the macromer composition and has at least two degradableregions attached to the core. At least two polymerizable regions areattached to the degradable regions so that, upon degradation, thepolymerizable regions, particularly in the polymerized gel form, areseparated. Conversely, if the central core of the macromer compositionis formed by a degradable region, at least two water soluble regions canbe attached to the core and polymerizable regions can be attached toeach water soluble region. The net result will be the same after gelformation and exposure to in vivo degradation conditions.

In another embodiment, the macromer composition has a water solublebackbone region and a degradable region affixed to the macromerbackbone. At least two polymerizable regions are attached to thedegradable regions, so that they are separated upon degradation,resulting in gel product dissolution. In a further embodiment, themacromer backbone is formed of a nondegradable backbone having watersoluble regions as branches or grafts attached to the degradablebackbone. Two or more polymerizable regions are attached to the watersoluble branches or grafts. In another variation, the backbone may bestar shaped, which may include a water soluble region, a biodegradableregion or a water soluble region which is also biodegradable. In thisgeneral embodiment, the star region contains either water soluble orbiodegradable branches or grafts with polymerizable regions attachedthereto. Again, the polymerizable regions must be separated at somepoint by a degradable region.

Polymerizable Groups

The polymerizable regions may be polymerizable by photoinitiation byfree radical generation, most preferably in the visible or longwavelength ultraviolet radiation. The preferred polymerizable regionsare acrylates, diacrylates, oligoacrylates, dimethacrylates,oligomethoacrylates, or other biologically acceptable photopolymerizablegroups. A preferred tertiary amine is triethanol amine.

Useful photoinitiators are those which can be used to initiate by freeradical generation polymerization of the macromers without cytotoxicityand within a short time frame, minutes at most and most preferablyseconds. Preferred dyes as initiators of choice for long wavelengthultraviolet (LWUV) light initiation are ethyl eosin,2,2-dimethoxy-2-phenyl acetophenone, other acetophenone derivatives,other eosin derivatives, such as eosin Y, and camphorquinone. In allcases, crossliking and polymerization are initiated among copolymers bya light-activated free-radical polymerization initiator such as2,2-dimethoxy-2-phenylacetophenone or a combination of ethyl eosin(10⁻⁴–10⁻² mM) and triethanolamine (0.001 to 0.1 M), for example.

The choice of the photoinitiator is largely dependent on thephotopolymerizable regions. For example, when the macromer includes atleast one carbon-carbon double bond, light absorption by the dye causesthe dye to assume a triplet state, the triplet state subsequentlyreacting with the amine to form a free radical which initiatespolymerization. Preferred dyes for use with these materials includeeosin dye and initiators such as 2,2-dimethyl-2-phenylacetophenone,2-methoxy-2-phenylacetophenone, and camphorquinone. Using suchinitiators, copolymers may be polymerized in situ by long wavelengthultraviolet light or by laser light of about 514 nm, for example.

Initiation of polymerization is accomplished by irradiation with lightat a wavelength of between about 200–700 nm, most preferably in the longwavelength ultraviolet range or visible range, 320 nm or higher, mostpreferably about 514 nm or 365 nm.

There are several photooxidizable and photoreducible dyes that may beused to initiate polymerization. These include acridine dyes, forexample, acriblarine; thiazine dyes, for example, thionine; xanthinedyes, for example, rose bengal; and phenazine dyes, for example,methylene blue. These are used with cocatalysts such as amines, forexample, triethanolamine; sulphur compounds, for example, RSO₂R₁;heterocycles, for example, imidazole; enolates; organometallics; andother compounds, such as N-phenyl glycine. Other initiators includecamphorquinones and acetophenone derivatives.

Thermal polymerization initiator systems may also be used. Such systemsthat are unstable at 37° C. and would initiate free radicalpolymerization at physiological temperatures include, for example,potassium persulfate, with or without tetramethyl ethylenediamine;benzoylperoxide, with or without triethanolamine; and ammoniumpersulfate with sodium bisulfite.

Other initiation chemistries may be used besides photoinitiation. Theseinclude, for example, water and amine initiation schemes with isocyanateor isothiocyanate containing macromers used as the polymerizableregions.

PREFERRED EMBODIMENTS

In a preferred embodiment, the polymeric materials in the macromercomposition are polymerizable and at least substantially water soluble.A first macromer includes at least one water soluble region, at leastone NO carrying region, and at least one free radical-polymerizableregion. A second macromer includes at least one water soluble region andat least two free radical polymerizable regions. The regions can, insome embodiments, be both water soluble and biodegradable. The macromercomposition is polymerized by exposure of the polymerizable regions tofree radicals generated, for example, by photosensitive chemicals anddyes.

Examples of these macromers are PVA or PEG. The choice of appropriateend caps permits rapid polymerization and gelation. Acrylates arepreferred because they can be polymerized using several initiatingsystems, e.g., an eosin dye, by brief exposure to ultraviolet or visiblelight. A PEG central structural unit (core) is preferred on the basis ofits high hydrophilicity and water solubility, accompanied by excellentbiocompatibility. A short oligo or poly(α-hydroxy acid), such aspolyglycolic acid, can be used as a biodegradable chain extensionbecause it rapidly degrades by hydrolysis of the ester linkage intoglycolic acid, a harmless metabolite. Although highly crystallinepolyglycolic acid is insoluble in water and most common organicsolvents, the entire macromer composition is water-soluble and can berapidly gelled into a biodegradable network while in contact withaqueous tissue fluids. Such networks can be used to entrap andhomogeneously disperse water-soluble drugs and enzymes and to deliverthem at a controlled rate. Further, they may be used to entrapparticulate suspensions of water-insoluble drugs. Other preferred chainextensions are polylactic acid, polycaprolactone, polyorthoesters, andpolyanhydrides. Polypeptides may also be used. Such “polymeric” blocksshould be understood to included dimeric, trimeric, and oligomericblocks.

PVA contains many pendant hydroxyl groups. These hydroxyl groups areeasily reacted to form side chains such as various crosslinking agentsand nitric oxide donors. PVA is water soluble and has excellentbiocompatiblity. Modification of PVA to attach methacrylate groups via adiacetal bond with the pendant hydroxyl groups and addition of anappropriate photoinitiator enables the PVA to be photopolymerized toform hydrogels under long wavelength UV light. In another preferredembodiment, the hydrogel is formed from modified polyvinyl alcohol (PVA)macromers, such as those described in U.S. Pat. Nos. 5,508,317,5,665,840, 5,849,841, 5,932,674, 6,011,077, 5,939,489, and 5,807,927.The macromers disclosed in U.S. Pat. No. 5,508,317, for example, are PVAprepolymers modified with pendant crosslinkable groups, such asacrylamide groups containing crosslinkable olefinically unsaturatedgroups. These macromers can be polymerized by photopolymerization orredox free radical polymerization, for example. Several embodiments ofthe macromers of the invention are disclosed herein describingformulations for photopolymerizable macromers. However, one of skill inthe art, after studying this disclosure, would know how to make and usemacromers formulated for other methods of polymerization. The startingpolymers are, in particular, derivatives of polyvinyl alcohol orcopolymers of vinyl alcohol that contain, for example, a 1,3-diolskeleton. The crosslinkable group or the further modifier can be bondedto the starting polymer skeleton in various ways, for example through acertain percentage of the 1,3-diol units being modified to give a1,3-dioxane, which contains a crosslinkable radical, or a furthermodifier in the 2-position. Another possibility is for a certainpercentage of hydroxyl groups in the starting polymer to be esterifiedby means of an unsaturated organic acid, these ester-bonded radicalscontaining a crosslinkable group. The hydrophobicity of these macromerscan be increased by substituting some of the pendant hydroxyl groupswith more hydrophobic substituents. The properties of the macromers,such as hydrophobicity, can also be modified by incorporating aco-monomer in the macromer backbone. The macromers can also be formedhaving pendant groups crosslinkable by other means.

B. NO Groups or Modulating Compounds

A number of molecules that produce NO under physiological conditions (NOdonors) have been identified and evaluated both in vitro and in vivo,including S-nitrosothiols, organic nitrates, and complexes of NO withnucleophiles. L-arginine is a NO donor, since L-arginine is a substratefor NO synthase, and thus administration of L-arginine increasesendogenous NO production and elicits responses similar to those causedby NO donors in most cases. Other NO donors include molsidomine, CAS754,SPM-5185, and SIN-1. Other compounds capable of producing and/ordonating NO may also be used. These include organic nitrates,nitrosylating compounds, nitrosoesters, and L-arginine.

The molecules which produce NO, or release or generate NO, arepreferably attached to regions containing nucleophiles and/or thiolssuch as S-nitrosothiols capable of forming a complex with NO.

C. Prophylactic, Therapeutic and Diagnostic Agents

The polymeric materials can also be used for drug delivery, preferablylocalized release of prophylactic, therapeutic or diagnostic agents atthe site where the materials are needed, although the polymericmaterials can be loaded with agent to be released systemically. Theseagents include proteins or peptides, polysaccharides, nucleic acidmolecules, and simple organic molecules, both natural and synthetic.Representative materials include antibiotics, antivirals, and antifungaldrugs, anti-inflammatories (steroidal or non-steroidal), hormones,growth factors, cytokines, neuroactive agents, vasoconstrictors andother molecules involved in the cardiovascular responses, enzymes,antineoplastic agents, local anesthetics, antiangiogenic agents,antibodies, drugs affecting reproductive organs, and oligonucleotidessuch as antisense oligonucleotides. Diagnostic materials may beradioactive, bound to or cleave a chromogenic substrate, or detectableby ultrasound, x-ray, MRI, or other standard imaging means.

These agents can be mixed with macromer prior to polymerization, appliedinto or onto the polymer, or bound to the macromer prior to or at thetime of polymerization, either covalently or ionically, so that theagent is released by degradation (enzymatic or hydrolytic) or diffusionat the site where the polymer is applied.

II. Methods of Use

A. Coatings; Films; Microparticles

Although described primarily with respect to in vivo treatment, it isapparent that the polymeric materials described herein can be used incell culture, on cell culture substrates, or as coatings on medicalimplants or devices such as stents, vascular grafts, or catheters, orformed using standard techniques into microparticles or other types offormulations which may be used in or administered to a patient.

For example, coatings on medical devices or implants may be used whenthe proliferation of endothelial cells over the medical device wouldprolong the use and safety of the device. One of skill in the art mayenvision many such uses. An example is found in dialysis. The coatingwould allow endothelial proliferation over the graft used during adialysis session, thereby prolonging the usable time for each graft,thus delaying the need for a kidney transplant. Coatings may provideother advantages seen upon local delivery of NO such as decreasedproliferation of smooth muscle cells and decreased platelet aggregation.

B. Therapeutic Applications

Polymeric materials capable of releasing physiological amounts of NO forprolonged periods of time can be applied to sites on or in a patient inneed of treatment thereof. Representative disorders or conditions thatcan be treated with NO include restenosis, thrombosis, asthma, woundhealing, arthritis, and penile or female erectile dysfunction. Thematerial can be applied as a macromer solution and polymerized in situor polymerization can be initiated prior to application. The polymericmaterials can also be coated onto medical devices.

Wound Healing

The formulations are particularly useful for treatment of all types ofwounds, including burns, surgical wounds, and open leg and foot wounds.There are generally three types of open leg wounds, termed ulcers:venous stasis ulcers, generally seen in sedentary elderly people whenblood flow to the leg becomes sluggish; decubitus ulcers, also termedpressure sores or bed sores, which occurs most often in people who arebedridden and are unable to frequently change position; and diabeticfoot ulcers, caused by poor blood circulation to the feet. Due to theaging of the population, there will likely be a greater demand foreffective and user friendly wound treatments in the near future.

The term “wound” as used herein refers to all types of tissue injuries,including those inflicted by surgery and trauma, including burns, aswell as injuries from chronic or acute medical conditions, such asatherosclerosis or diabetes.

Example 13 shows that exogenous NO released from hydrogel wounddressings may enhance wound healing of chronic wounds. In vivo resultsexamining effects of NO in the diabetic wound model suggest that themost useful parameters for assessing efficacy of wound healing aregranulation tissue thickness and matrix production. Similar findingshave been reported in studies examining the effect of growth factors onwound healing in animals (Greenhalgh D. The role of growth factors, inwound healing. J Trauma 1996; 41: 159–167). A review of multiple animalmodels for assessing efficacy of PDGF concluded that epithelializationand wound contraction were not significantly altered, whereas in mostmodels, including the diabetic mouse model, granulation tissue thicknesswas consistently increased following application of the growth factor(LeGrand E K. Preclinical promise of Becaplermin (rhPDGF-BB) in woundhealing. Am J Surg 1998; 176:48S–54S).

Similar improvements in wound healing appear to occur followingapplication of NO, and this might be attributed to the inter-relatedmechanisms of action of NO with growth factors.

The significant increase in in vivo wound collagen deposition caused bytreatment with NO indicates that delivery of NO from PVA hydrogel wounddressings may lead to the development of a more structurally stableclosed wound. This finding is important, as chronic wounds arefrequently complicated by their inability to remain healed due toinsufficient mechanical integrity.

NO clearly plays a critical role in the wound healing process, mostprobably via multiple mechanisms including increased cell proliferationvia upregulation of growth factor receptors and upregulation of matrixsynthesis. That growth factors alone also enhance wound healing suggeststhat combining NO with growth factors may lead to synergistic effects.

Hydrogels may be modified to covalently attach growth factors, whilemaintaining the bioactivity of the growth factor (Mann B, Schmedlen R,West J. Tethered-TGF-beta increases extracellular matrix production ofvascular smooth muscle cells. Biomaterials 2001; 22: 439–444). Thus, itis possible to develop hydrogels that provide combined NO and growthfactor therapy to further enhance the healing of chronic wounds.

The materials described herein overcome some of the disadvantages ofexisting wound treatments by allowing the formation of hydrogel coatingsthrough in situ polymerization. This technology may simplify the oftendifficult application of wound dressings to areas such as foot ulcers.Additionally, a multitude of factors to promote wound healing may beincorporated into these dressings through simple modification of thehydrogel material.

Treatment of Restenosis

A preferred application is a method of reducing the effects ofrestenosis on post-surgical patients. One embodiment of the methodincludes coating the surface within an artery with an aqueous solutionof light-sensitive free radical polymerizable initiator and a number ofmacromers. The coated artery is subjected to a Xenon arc laser inducingpolymerization of the macromers. As the newly polymerized macromercomposition is formed, the physiological conditions within the arterywill induce the release of NO. This release will be strictly localizedfor prolonged periods of time. In another embodiment of the method, astent coated with the NO-releasing hydrogel is implanted in an artery.

Prevention of Surgical Adhesions.

A preferred application is a method of reducing formation of adhesionsafter a surgical procedure in a patient. In one embodiment the methodincludes coating damaged tissue surfaces in a patient with an aqueoussolution of a light-sensitive free-radical polymerization initiator anda macromer solution as described above. The coated tissue surfaces areexposed to light sufficient to polymerize the macromer. Thelight-sensitive free-radical polymerization initiator may be a singlecompound (e.g., 2,2-dimethoxy-2-phenyl acetophenone) or a combination ofa dye and a cocatalyst (e.g., ethyl eosin and triethanol amine).

Tissue Adhesives.

Another use of the polymers is in a method for adhering tissue surfacesin a patient. In one embodiment the macromer is mixed with aphotoinitiator or photoinitiator/cocatalyst mixture to form an aqueousmixture and the mixture is applied to a tissue surface to which tissueadhesion is desired. The tissue surface is contacted with the tissuewith which adhesion is desired, forming a tissue junction. The tissuejunction is then irradiated until the macromers are polymerized.

Tissue Coatings

In a particularly preferred application of these macromers, an ultrathincoating is applied to the surface of a tissue, most preferably the lumenof a tissue such as a blood vessel. One use of such a coating is in thetreatment or prevention of restenosis, abrupt reclosure, or vasospasmafter vascular intervention. An initiator is applied to the surface ofthe tissue, allowed to react, adsorb or bond to tissue, the unboundinitiator is removed by dilution or rinsing, and the macromer solutionis applied and polymerized. This method is capable of creating uniformpolymeric coating of between one and 500 microns in thickness, mostpreferably about twenty microns, which does not evoke thrombosis orlocalized inflammation.

Tissue Supports

The polymeric materials can also be used to create tissue supports byforming shaped articles within the body to serve a mechanical function.Such supports include, for example, sealants for bleeding organs,sealants for bone defects and space-fillers for vascular aneurisms.Further, such supports can include strictures to hold organs, vessels ortubes in a particular position for a controlled period of time.

Controlled Drug Delivery

As noted above, the polymeric materials can be use as carriers forbiologically active materials such as therapeutic, prophylactic ordiagnostic agents, including hormones, enzymes, antibiotics,antineoplastic agents, and cell suspensions. The polymeric material maybe used to temporarily preserve functional properties of an agent to bereleased, as well as provide prolonged, controlled release of the agentinto local tissues or systemic circulation.

In a variation of the method for controlled drug delivery in which anagent is mixed with the macromer solution then polymerized in situ, themacromers are polymerized with the biologically active materials to formmicrospheres or nanoparticles containing the biologically activematerial. The macromer, photoinitiator, and agent to be encapsulated aremixed in an aqueous mixture. Particles of the mixture are formed usingstandard techniques, for example, by mixing in oil to form an emulsion,forming droplets in oil using a nozzle, or forming droplets in air usinga nozzle. The suspension or droplets are irradiated with a lightsuitable for photopolymerization of the macromer.

These materials are particularly useful for controlled drug delivery ofhydrophilic materials, since the water soluble regions of the polymerenable access of water to the materials entrapped within the polymer.Moreover, it is possible to polymerize the macromer compositioncontaining the material to be entrapped without exposing the material toorganic solvents. Release may occur by diffusion of the material fromthe polymer prior to degradation and/or by diffusion of the materialfrom the polymer as it degrades, depending upon the characteristic poresizes within the polymer, which is controlled by the molecular weightbetween crosslinks and the crosslink density. Deactivation of theentrapped material is reduced due to the immobilizing and protectiveeffect of the gel and catastrophic burst effects associated with othercontrolled-release systems are avoided. When the entrapped material isan enzyme, the enzyme can be exposed to substrate while the enzyme isentrapped, provided the gel proportions are chosen to allow thesubstrate to permeate the gel. Degradation of the polymer facilitateseventual controlled release of free macromolecules in vivo by gradualhydrolysis of the terminal ester linkages.

As demonstrated by examples 1–3 below, three classes of NO-producing,PEG-based polymers have been synthesized and their NO release rateconstants determined in vitro under physiological conditions. Thebiological response to appropriate materials has been evaluated in vitrousing cultured smooth muscle cells and endothelial cells and in vivousing a rat carotid artery injury model that resembles restenosis inman. The materials include BAB block copolymers of polyethylene glycol(A) with polycysteine (B) that are subsequently reacted with NaNO₂ toform S-nitrosothiols, BAB block copolymers of polyethylene glycol(“PEG”) (A) and diethylenetriamine (“DETA”) (B) that are subsequentlyreacted with NO gas to form nucleophile/NO complexes, and BAB blockcopolymers of polyethylene glycol (A) and polylysine (B) that aresubsequently reacted with NO gas to form nucleophile/NO complexes.Blended compounds may also be prepared for providing biphasic release ofprofiles, such as PEG-Cys-DETA-NO. All polymers are further terminatedwith reactive acrylate groups to allow rapid polymerization in situ.

Such materials would be expected to have good biocompatibility, providedthat a water soluble, biocompatible polymer such as PEG comprises thebulk of the material and has a sufficiently high molecular weight, andto slowly biodegrade due to the presence of two ester bonds and twoamide bonds in each polymer chain. These three materials were selectedas they are expected to have vastly different release kinetics:nucleophile/NO complexes have been shown to release NO for up to 5 weeks(Smith el al., (1996) J. Med. Chem., 39:1148–56), while the half-life ofS-nitrosocysteine is 0.023 hours (Mathews et al., (1993) J. Pharmacol.Exp. Therap., 267:1529–37). The amount of NO produced by thesecopolymers may be tailored by altering the ratio of polyethylene glycol(PEG) to cysteine or lysine.

An advantage of these macromer compositions are that they can bepolymerized rapidly in an aqueous surrounding. Precisely conforming,semi-permeable, biodegradable films or membranes can thus be formed ontissue in situ to serve as biodegradable barriers, as carriers forliving cells or other biologically active materials, and as surgicaladhesives. The polymer shows excellent biocompatibility, as seen by aminimal fibrous overgrowth on implanted samples. Hydrogels for themodels were gelled in situ from water-soluble precursors by briefexposure to LWUV light, resulting in formation of an interpenetratingnetwork of the hydrogel with the protein and glycosaminoglycancomponents of the tissue.

As demonstrated by examples 4 and 5 below, three types of PVA hydrogelswere made and demonstrated release of NO and incorporated drug (bFGF):PVA-NH₂—NO hydrogels; PVA-Cys-NO hydrogels; PVA-NO-bFGF hydrogels. Theresults are similar to those for the PEG based hydrogels.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are intended neither to limit nor define the invention in anymanner.

EXAMPLES Example 1 Synthesis of PEG-Cys-NO Macromers and Hydrogels

As shown in FIG. 1, an acryloyl-PEG-Cys-NO polymer was formed by firstreacting polyethylene glycol N-hydroxysuccinimide monoacrylate(ACRL-PEG-NHS, MW 3400, commercially available from Shearwater Polymers,Huntington, Ala.) with L-cysteine at an 1:2 molar ratio in 50 mM sodiumbicarbonate buffer (pH 8.5) for 2 hours; the product was then dialyzedin a cellulose ester membrane (Molecular weight cutoff 500, SpectrumLabs, Laguna Hills, Calif.) in diH₂O, and lyophilized. Analysis of theacryloyl-PEG-Cys copolymer was performed using gel permeationchromatography (GPC) with an evaporative light scattering detector and aUV detector at 260 nm (Polymer Laboratories, Amherst, Mass.). Successfulsynthesis of acryloyl-PEG-Cys was determined by a shift in the positionof the peak from the evaporative light scattering detector. Thecopolymer was then reacted with an equimolar amount of NaNO₂ at pH 2 and37° C. for 20 minutes to form S-nitrosocysteine. Conversion of thiolgroups to S-nitrosothiols was measured using the Ellman's assay(Hermanson, (1995) Bioconjugate Techniques, San Diego, Calif. AcademicPress; 88–90). After adjusting the pH of the solution to 7.4, theacryloyl-PEG-Cys-NO polymer was incorporated into photopolymerizablehydrogels by mixing with PEG-diacrylate (MW 3400) at a 1:10 molar ratioin aqueous solution with 1500 ppm 2,2-dimethoxy-2-phenyl acetophenone asa long wavelength ultraviolet initiator. 0.15% N-vinylpyrrolidone waspresent in this mixture as it was used as a solvent for thephotoinitiator. Exposure to UV light (365 nm, 10 mW/cm²) was used tocrosslink the polymer, resulting in conversion to a hydrogel (Sawhney etal., (1993) Macromol 26:581–7).

Example 2 Synthesis of PEG-Lys₅-NO Macromers and Hydrogels

As shown in FIG. 2, for acryloyl-PEG-Lys₅-NO hydrogels, a copolymer ofACRL-PEG-NHS (MW 3400, Shearwater Polymers) and poly-L-lysine (DP=5) wassynthesized by reacting at an equimolar ratio in 50 mM sodiumbicarbonate (pH 8.5). The resultant copolymer was analyzed via GPC, thendissolved in water and reacted with NO gas in an evacuated vessel, thusforming NO-nucleophile complexes with the amine groups on the lysineside groups. The extent of conversion of amine groups to NO-nucleophilecomplexes was measured using the ninhydrin assay, and crosslinkedhydrogels were formed as described above in Example 1.

Example 3 Synthesis of PEG-DETA-NO Macromers and Hydrogels

Diethylenetriamine (DETA, Aldrich, Milwaukee, Wis.) was reacted withACRL-PEG-NHS (MW 3400, Shearwater Polymers) in 50 mM sodium bicarbonatebuffer (pH 8.5) at an equimolar ratio, lyophilized, and analyzed via GPCas described above. The copolymer was then dissolved in water andexposed to NO gas to form NO-nucleophile complexes as described forPEG-Lys₅-NO and assayed for amine content using the ninhydrin assay. ThePEG-DETA-NO was lyophilized and then photopolymerized as described aboveto form hydrogels, as shown in FIG. 3.

Example 4 Synthesis of PVA-NH₂—NO Macromers and Hydrogels

Poly(vinyl alcohol) (Hoechst, Mowiol 4-88) was dissolved in diH₂0 andwarmed to 95° C. in a round bottom flask under continuous stirring.After one hour, the solution was cooled to room temperature, and acrosslinkable acetal group, methacrylamidoacetaldehyde dimethyl acetal(NAAADA) was added. The amine acetal, gamma-aminobutyraldehyde diethylacetal, was also added, and the mixture was acidified using glacialacetic acid and 37% hydrochloric acid. The mixture was allowed to stirat room temperature for nine hours, after which the pH was adjusted topH 3.6 using triethylamine. In order to purify the polymer, the solutionwas then diafiltered through a MW 3000 cellulose membrane against diH₂Oat 6.5 times the volume of polymer solution. The polymer concentrationwas adjusted to 22% w/v using diafiltration, and the pH was adjusted to7.4 with 1N NaOH. The amine concentration of the polymer was determinedusing the ninhydrin assay.

In order to form the NO donor bound to the PVA-NH₂, the neutralizedamine-modified polymer was placed in a round bottom flask with stopcock.The flask was evacuated and filled with nitric oxide gas until thedesired conversion of amines to NO nucleophile complexes was achieved.Photocrosslinked hydrogels were formed from the PVA-NH₂—NO by adding0.1% IRGACURE™ 2959 (Ciba-Geigy) photoinitiator (based on total solutionvolume) and then exposing to UV light (2 mW/cm², 365 nm) for 30 seconds.Addition of the photoinitiator brings the final polymer concentration to20% w/v.

Example 5 Synthesis of PVA-Cys-NO Macromers and Hydrogels

PVA-NH₂ was synthesized as described above. The amine terminus ofcysteine was acetylated using acetic anhydride, and the carboxyl end ofthe cysteine was coupled to the PVA-NH₂ using water-soluble EDACchemistry. The resulting PVA-Cys was then purified using diafiltrationand brought to a concentration of 22% w/v. PVA-Cys-NO was formed byadding sodium nitrite at an equimolar amount to cysteine residues,adjusting the pH to 2, and incubating at 37° C. for 15 minutes. Theextent of reaction of cysteine to Cys-NO was assayed using both theEllman's and Griess reactions. The photoinitiator,2,2-methyl-2-phenylacetophenone was dissolved in N-vinylpyrrolidone at aconcentration of 600 mg/ml and added to the polymer solution (0.1% basedon total solution volume). The polymer was then crosslinked under UVlight for 30 seconds and placed in HEPES buffered saline, pH 7.4, 37° C.

Example 6 Synthesis of PVA-NO-bFGF Hydrogels

For PVA-NO-bFGF hydrogels, the above procedure was used to make thePVA-NO polymer. Immediately prior to exposure to UV light, 25 μg/ml bFGFwas added to the polymer solution and mixed well. Gels were crosslinkedas described earlier and stored in HEPES buffered saline, pH 7.4, 37° C.

Example 7 NO Release from Hydrogels

Following preparation and polymerization of the NO-releasing materialsas described above, the hydrogels were weighed and stored in HEPESbuffered saline, pH 7.4, at 37° C. Aliquots of the buffer were removedat each time point and replaced with fresh buffer. The samples from eachtime point were then analyzed for nitrite content using a colorimetricassay based on the Griess reaction.

NO release kinetics of hydrogels stored in buffer at various pH levelswere also investigated in order to explore possible storage conditionsfor the hydrogels. At acidic pH levels, release of NO from the hydrogelswas significantly inhibited.

NO release from acryloyl-PEG-Lys₅-NO hydrogels is shown in FIG. 4.

NO release from acryloyl-PEG-DETA-NO hydrogels is shown in FIG. 5.

NO release from acryloyl-PEG-Cys-NO hydrogels is shown in FIG. 6.

Example 8 NO and bFGF Release from PVA-NO-bFGF Hydrogels

The release of NO release from PVA-NO-bFGF hydrogels prepared asdescribed in Example 6 was determined in the same manner as Example 7and is shown in FIG. 7A. Release of bFGF was quantified using that BCAassay (Pierce Chemicals) and is shown in FIG. 7B. Release of NOcontinues for well over 12 hours, while the growth factor is completelyreleased within the first 5 hours.

Example 9 Effects of NO-Releasing Macromers on Cultured Smooth MuscleCells Proliferation and Viability

In order to assess the potential of a material for the reduction ofsmooth muscle cell proliferation after vascular injury, cultured smoothmuscle cells were grown in the presence of NO-releasing materials, andthe effects of those materials on the cells evaluated. Smooth musclecells isolated from Wistar-Kyoto rats (passage 11–15, provided by T.Scott-Burden) were cultured in Minimum Essential Medium supplementedwith 10% FBS, 2 mM L-glutamine, 500 units penicillin, and 100 mg/Lstreptomycin, at 37° C. in a 5% CO₂ environment. The cells were seededinto 24-well tissue culture plates (Becton Dickinson, Franklin Lakes,N.J.) at a density of 10,000 cells/cm². NO donors in either soluble orhydrogel form were added to the media in the wells one day afterseeding. At 4 days culture, cell numbers were determined by preparingsingle cell suspensions with trypsin and counting three samples fromeach group using a Coulter counter (Multisizer #0646, CoulterElectronics, Hialeah, Fla.).

The effects of NO donors in solution on the proliferation of smoothmuscle cells were first investigated by performing a NO dose responsecurve, whereupon cells were cultured with a range of NO donorconcentrations (1 μM–10 mM) in order to identify appropriate dosages forhydrogel studies. NO-nucleophile complexes (Lys-NO and DETA-NO) wereformed by reacting either L-lysine or DETA with NO gas in water for 24hours. Soluble Cys-NO was synthesized by reacting an equimolar amount ofL-cysteine with NaNO₂ at pH 2 and 37° C. for 20 minutes. All NO donorsolutions were adjusted to pH 7.4 prior to addition to cell cultures.

Smooth muscle cell proliferation in the presence of NO-producing andcontrol hydrogels was then investigated using the optimal NO dosedetermined above. Hydrogels containing acryloyl-PEG-Lys₅-NO,acryloyl-PEG-DETA-NO, and acryloyl-PEG-Cys-NO were formed as describedabove, except that the gel solutions were sterile filtered through 0.2μm syringe filters (Gelman Sciences, Ann Arbor, Mich.) prior to adding2,2-dimethoxy-2-phenyl acetophenone. PEG-diacrylate hydrogels containingno NO donors were used as a control. The hydrogels were photopolymerizedin cell culture inserts (8 μm pore size, Becton Dickinson, FranklinLakes, N.J.) and placed in the media over the cultured cells.

All three hydrogel NO donors significantly inhibited SMC growth(p<0.0001). The number of smooth muscle cells remained near that of theseeding density, which ranged from 10–15% of the final control cellnumber for all experiments.

Inhibition of SMC proliferation by acryloyl-PEG-Lys₅-NO hydrogels isshown in FIG. 8A, compared to the macromer solution control shown inFIG. 8B. Both significantly inhibited SMC proliferation.

Inhibition of SMC proliferation by acryloyl-PEG-DETA-NO-nucleophilecomplex hydrogels is shown in FIG. 9A, compared to the macromer solutioncontrol shown in FIG. 9B. Both significantly inhibited SMCproliferation.

Inhibition of SMC proliferation by acryloyl-PEG-Cys-NO hydrogels isshown in FIG. 10A, compared to the macromer solution control shown inFIG. 10B. Both significantly inhibited SMC proliferation.

Inhibition of SMC proliferation by acryloyl-PEG-Cys-NO hydrogels,acryloyl-PEG-DETA-NO hydrogels, and acryloyl-PEG-Lys-NO hydrogels iscompared to the control hydrogel in FIG. 11. All of the NO hydrogelssignificantly inhibited SMC growth.

Example 10 Effects of NO-Releasing Macromers on Platelet Adhesion InVitro

The effect of NO release on platelet adhesion was investigated to assessthe potential of these materials for prevention of thrombosis. Blood wasobtained from a healthy volunteer by venipuncture and anticoagulatedwith 10 U/ml heparin. Platelets and white blood cells were fluorescentlylabeled with mepacrine at a concentration of 10 μM. A solution of 2.5mg/ml collagen I in 3% glacial acetic acid in diH₂O was prepared andapplied to glass slides for 45 minutes in a humidified environment atroom temperature. Acryloyl-PEG-Cys-NO and PEG-diacrylate hydrogels wereprepared as described above and incubated with the labeled whole bloodat 37° C. for 30 minutes. The hydrogels were removed and the blood wasthen incubated with the collagen-coated glass slides (two per group) for20 minutes at 37° C. and then rinsed with HBS. Platelet counts per fieldof view at 40×were counted under a fluorescent microscope (ZeissAxiovert 135, Thornwood, N.Y.) in four randomly chosen areas per slide.

Photos of platelets which had been exposed to control PEG-diacrylate oracryloyl-PEG-Cys-NO hydrogels demonstrate that exposure to theNO-releasing hydrogels inhibits platelet adhesion to thrombogenicsurfaces. Glass slides coated with collagen were used as a thrombogenicsurface to which platelets would normally adhere. When the blood wasincubated with control PEG-diacrylate hydrogels, 69.25±4.46 (mean±SD)adherent platelets were observed per field of view. This number wasreduced to 7.65±6.16 platelets pre field of view when blood waspre-exposed to the acryloyl-PEG-Cys-NO hydrogels p<0.0001).

Example 11 Effects of NO-Releasing Macromers on Cultures EndothelialCells Proliferation and Viability

The polymeric materials described herein can be used to increaseproliferation of endothelial cells.

Cell Viability

Bovine aortic endothelial cells (BAECs, passage 5–10, Clonetics) werecultured in Dulbecco's Modified Eagle Medium with identical supplementsand culture conditions to the smooth muscle cells. The viability ofendothelial cells exposed to NO-releasing PEG gels was examined throughthe use of a Live/Dead staining kit (Molecular Probes, Eugene, Oreg.).BAECs were seeded into 24-well plates at a concentration of 10,000cells/cm², and hydrogels were added as described above. After two daysin culture, cell viability was assessed. As discussed earlier, a 4 μMsolution of ethidium bromide causes dead cells to fluoresce red due totheir increased permeability, while a 2 μM solution of calcein AM causesviable cells to fluoresce green due to esterase activity. Cells wereexamined under a fluorescence microscope (Zeiss Axiovert 135, Thornwood,N.Y.), and photomicrographs were taken using a digital camera (Sony).

Endothelial Cell Proliferation

BAECs cells were seeded into 24-well tissue culture plates (BectonDickinson, Franklin Lakes, N.J.) at a density of 10,000 cells/cm². NOdonors in soluble form (1 μM–10 mM) were added to the media in the wellsone day after seeding. After 4 days of culture, cell numbers weredetermined by preparing single cell suspensions with trypsin andcounting three samples from each group using a Coulter Counter(Multisizer #0646, Coulter Electronics, Hialeah, Fla.).

Endothelial cell proliferation in the presence of NO-producing andcontrol hydrogels was then investigated using the optimal NO dosedetermined above. After four days in culture with the hydrogels, cellnumbers were determined by preparing single cell suspensions withtrypsin and counting three samples from each group using a CoulterCounter as described above.

Endothelial Cell Proliferation on NO-Releasing Hydrogels

In order for these hydrogels to effectively prevent restenosis,re-endothelialization must occur not only in areas surrounding thehydrogel, but also upon the hydrogel itself. To investigate theproliferation of endothelial cells cultured on NO-releasing hydrogels, acell adhesion ligand was first covalently incorporated into thesehydrogels, as cells will not attach to PEG unless the polymer ismodified with an adhesive sequence (Hem D, Hubbell J. Incorporation ofadhesion peptides into nonadhesive hydrogels useful for tissueresurfacing, J Biomed Mater Res 1998; 39: 266–276). To achieve this,hydrogels containing the adhesive peptide sequence RGDS(Arginine-Glycine-Aspartic acid-Serine) and the NO donor DETA-NO weresynthesized. RGDS was covalently bound to PEG by reaction withACRL-PEG-NHS in a ratio of 1:2 (RGDS:polymer) in 50 mM sodiumbicarbonate buffer (pH 8.5) for two hours at room temperature. Thesolution was then dialyzed and lyophilized to obtain ACRL-PEG-RGDS.ACRL-PEG-DETA-NO was prepared as described above. These two copolymerswere blended with PEG-diacrylate to achieve a final RGDS concentrationof 1.4 μmol/ml of polymer, and 1.25 μmol DETA-NO/ml of polymer, whichwould theoretically deliver a total of 50 nmol NO donor per ml of cellculture media. The hydrogel precursor solution was filter sterilized andpoured between two polystyrene plates separated by a 400 μm gap. Thehydrogel precursor was exposed to UV light, and a sterilized cork-borerpunch (Cole Parmer, Vernon Hills, Ill.) was used to create thin,circular hydrogels that were subsequently placed in a 24-well plate.BAECs were immediately seeded upon the hydrogels at a density of 7500cells/cm². Controls consisted of hydrogels with the NO donor alone orRGDS alone. Two days after cell seeding, cells were trypsinized and cellnumber was assessed by counting on a Coulter Counter.

Hydrogels containing the cell adhesive ligand REDV were also prepared.Synthesis of acryloyl-PEG-REDV was identical to the synthesis ofacryloyl-PEG-RGDS except that the concentration of REDV in the hydrogelswas 14 μmol/ml. Endothelial cell seeding experiments were alsoidentical.

Results: Cell Proliferation and Viability

The effects of NO release from these hydrogel materials on endothelialcell proliferation was investigated in order to examine whether NOdelivery would have a stimulatory effect on re-endothelializationfollowing vascular injury. A concentration of 0.5 μmoles NO released in1 ml media (0.5 mM) over the course of the experiment was chosen as theoptimal NO concentration from studies performed with a range of solubleNO donor concentrations; this concentration resulted in increasedproliferation of endothelial cells. At lower concentrations of NO (50μM–0.1 mM), endothelial cell proliferation was still significantlyincreased, while concentrations greater than 1 mM resulted in a decreasein cell number. All three hydrogel NO donors significantly increasedendothelial cell growth with no change in cell viability (FIG. 12),indicating that release of NO from PEG hydrogels may lead to fasterre-endothelialization of vessels following injury, which would bebeneficial in preventing restenosis.

Endothelial Cell Proliferation on NO-Releasing Hydrogels

Hydrogels were synthesized with the covalently bound adhesive peptidesequence RGDS in addition to the NO donor DETA-NO in order to examineendothelial cell proliferation when cultured on NO-releasing hydrogels.After two days in culture, there were significantly more cells on thehydrogels containing both RGDS and DETA-NO than on either of the controlmaterials (FIG. 13). As expected, very few cells were adhered to thehydrogels containing DETA-NO with no peptide sequence. While cellsattached and proliferated on the hydrogels containing RGDS but no NOdonor, the combination of the NO donor with the peptide sequence allowedfor increased proliferation over the peptide alone. These findingsfurther illustrate the ability of NO to stimulate endothelial cellproliferation. Additionally, this experiment demonstrates that moleculessuch as peptide sequences may also be covalently bound to NO-releasinghydrogels in order to design a material that further encouragesre-endothelialization. Potential applications for these materials arenot limited to the prevention of restenosis, as they may also be used tocoat blood-contacting devices such as stents or vascular grafts whereenhanced endothelialization is desirable.

Endothelial Cell Proliferation Employing Cell Adhesion Ligand REDV

Hydrogels containing the cell adhesive ligand REDV were also prepared.Synthesis of acryloyl-PEG-REDV was identical to the synthesis ofacryloyl-PEG-RGDS except that the concentration of REDV in the hydrogelswas 14 μmol/ml and the concentration of RGDS was 1.4 μmol/ml.Endothelial cell seeding experiments were also identical. FIG. 14 showsthe average cell number data for acryloyl-PEG-REDV.

Example 12 NO-Producing PEG Derivations Combined with PEG-Diacrylate

The monoacrylate NO-producing PEG derivatives have been combined withPEG-diacrylate to allow crosslinking into hydrogels. BiodegradablePEG-diacrylate derivatives, such as copolymers with α-hydroxy acids(Sawhney A S, Pathak C P, Hubbell J A. Bioerodible hydrogels based onphotopolymerized poly(ethylene glycol)-co-poly(alpha-hydroxy acid)diacrylate macromers. Macromol 1993; 26: 581–587) or proteolyticallydegradable peptides (West J L, Hubbell J A. Polymeric biomaterials withdegradation sites for proteases, involved in cell migration. Macromol1999; 32: 241–244) could be substituted for PEG-diacrylate to createbiodegradable, NO-producing hydrogels. This allows separatedetermination of NO production kinetics and biodegradationcharacteristics. Flexibility in the duration of NO release may alsoprove useful in the extension of this therapy to applications other thanthrombosis and restenosis.

Example 13 Evaluation of Hydrogels for Enhancement of Wound Healing

In Vitro Evaluation of Effects of PVA-NO on Fibroblast Viability andProliferation

Human dermal fibroblasts (HDFs, passage 5–11, Clonetics) were culturedin Dulbecco's Modified Eagle Medium supplemented with 10% FBS, 2 mML-glutamine, 500 units penicillin, and 100 mg/L streptomycin, at 37° C.in a 5% CO₂ environment. The effects of NO release from PVA-NO hydrogelson the viability and growth of HDFs were investigated in in vitrostudies. HDFs were seeded at 8000 cells/cm² in 24-well tissue culturepolystyrene plates. NO-releasing hydrogels containing 0.1 μmol to 5 μmolof the NO donor(0.1 mM to 5 mM NO donor in 1 ml cell culture media) werepolymerized and suspended in the cell culture media in transwell inserts24 hours following cell seeding. The hydrogels release NO over a periodof approximately two days. Viability after 4 days of culture wasassessed using trypan blue dye exclusion. After 4 days of culture, thecells were counted using a Coulter Counter to assess the effect of NOrelease on cell proliferation.

In Vitro Evaluation of the Effects of PVA-NO on Extracellular MatrixProduction

Extracellular matrix (ECM) production was assessed in fibroblast culturethrough incorporation of ³H-glycine into glycoprotein, elastin, andcollagen portions of the ECM as determined by sequential enzymedigestion (TEC assay; Scott-Burden T, Resink T, Bürgin M, Bühler F.Extracellular matrix: Differential influence on growth and biosynthesispatterns of vascular smooth muscle cells from SRI and WKY rats. J CellPhysiol 1989; 141: 267–274). HDFs were seeded at 8000 cells/cm² in24-well tissue culture polystyrene plates, and PVA-NO hydrogels wereformed in transwell inserts and added to the cell culture media 24 hoursfollowing cell seeding. The media was supplemented with 1 μCi/ml³H-glycine at this time. The same procedure was followed for cellsintended for counting, except that the media was not supplemented with³H-glycine. Two days following the addition of the hydrogels, the cellsin non-radioactive wells were trypsinized and counted on a CoulterCounter. The cells in the remaining wells were lysed in a solution of 25mM ammonium hydroxide for 30 minutes, and the plate was then dehydrated.A sequential digestion of extracellular matrix was performed in order todigest glycoproteins, elastin, and collagen. Radioactivity in samplesfrom each digestion step was determined by scintigraphy (MinaxiβTri-Carb 4000, Packard Instrument Co., Meridien, Conn.).

In Vivo Evaluation of PVA-NO Hydrogels for Enhancement of Wound Healing

The animal model used for in vivo testing of the PVA-NO hydrogels was afull thickness wound (1.5 cm diameter) in the dorsal skin of geneticallydiabetic (C57BLK/J-m+/+/Lepr^(db)) female mice, 8 weeks of age (JacksonLabs., Bar Harbor, Minn.). A total of 21 mice were used with equalnumbers being assigned to each of 3 groups (2 test groups and 1 controlgroup). Using the results from in vitro studies as well as data from theliterature, the doses of NO selected for in vivo studies were set at 0.5and 5 mM. These concentrations correspond to a total of 0.5 μmol and 5μmol, respectively, of NO released from the hydrogels over a period ofapproximately 30 hours. Test groups had either 0.5 mM or 5 mM hydrogelsapplied and control group were dressed with PVA hydrogel without NOadded. At time=0 days, a full thickness, 1.5 cm diameter wound wascreated. Mice were anesthetized by isoflurane inhalation and the dorsalskin was prepared for surgery using Betadine and 70% isopropanol. A fullthickness, 1.5 cm diameter wound was created by surgical excision ofepidermal and dermal layers. A circular piece of sterile PVA-NO orcontrol hydrogel was cut to match the size of the wounds using a sterilecircular cork borer, and applied to the wound. The wounds were coveredwith a transparent semi-occlusive secondary dressing (Tegaderm, 3M),adhered to the area surrounding the wound using tincture of Benzoin.

Every 2 to 3 days following surgery, wounds were redressed while themice were under isoflurane inhalation anesthesia. The secondary dressingand the hydrogel were removed and the wounds were flushed with sterilesaline to remove debris and to clean the wound area. A digitalplanimetric image of the wound was recorded using a Pixera video camera.A calibration scale was recorded with each image. Once photographed,fresh dressings were placed on the wounds, and the wounds were coveredagain with fresh Tegaderm dressings.

Wound area was assessed by image analysis using ImagePro Plus 3.0 imageanalysis software. Using the acquired images and this software, theperimeter of the wound was defined and measured, and the wound areasdetermined. Means and standard deviations of wound perimeters and areasat each time point were calculated.

One animal from each group was sacrificed at each time point of 8, 15and 22 days and the final 4 animals from each group were sacrificed at29 days. Histology was conducted on one animal from each group at eachtime point. Tissue surrounding and underlying the wound was sampled fromthese mice at the time of sacrifice and was fixed in Streck tissuefixative (Zinc-formalin), and embedded in paraffin for histologicalsectioning. Sections from each wound were stained with Hematoxylin andEosin and with Masson's Trichrome stains. Granulation tissue thicknesswas measured at days 8 and 15 and collagen layer thickness was measuredin sections from the final time point (29 days). Tissue thickness wasmeasured using ImagePro Plus 3.0 image analysis software on imagescaptured using an Olympus BX50WI microscope and SONY DKC 5000 camera.

Control of bias was achieved by assigning a color code to each of thetest groups and the control group. Investigators were blinded to theidentity of each of the groups and the test and control hydrogels have asimilar appearance. All animal experimentation was conducted underappropriate procedures approved by the University of Medicine andDentistry of New Jersey animal care and use review boards.

Characterization of Release Kinetics

As shown previously, release of NO from PVA-NO hydrogels was observedover a period of 48 hours at pH 7.4, as determined by the Griess assay.A slightly acidic pH is often observed in the wound environment, causingus to also evaluate NO release from these hydrogels at pH 6. Noinhibition of NO release was observed when hydrogels were exposed toslightly acidic conditions. Hydrogels may be tailored to obtain a rangeof NO concentrations by blending with unreacted aminated PVA prior topolymerization, allowing easy tailoring of the NO dosage. Controlhydrogels for release kinetics studies consisted of aminated PVA whichhad not been exposed to NO, as well as unmodified PVA (no amine groups)which had been exposed to NO, but did not contain any reactive groupswith which to form NO donors. No significant NO release was observedwith either control group.

In Vitro Evaluation of Effects of PVA-NO on Cell Viability,Proliferation, and ECM Synthesis

Exposure of HDFs to range of concentrations of PVA-NO hydrogels did notaffect cell viability, as measured by trypan blue exclusion. Cells inall conditions remained >90% viable, even at the highest NOconcentration of 5 mM. There was also no change in cell proliferation,as measured by cell counts (FIG. 14).

Analysis of ECM synthesis by HDFs cultured in the presence of PVA-NOhydrogels for two days indicated increased collagen production withincreasing NO concentration (FIG. 15A; p<0.01). There was also a slightincrease in overall matrix production by cells exposed to 5 mM PVA-NO,although this difference was not significant (FIG. 15B).

In Vivo Evaluation of PVA-NO Hydrogels for Enhancement of Wound Healing

Wound closure first occurred by day 15 in one of 6 remaining animals inthe control group. At day 22, 2/5 animals remaining in both the controland the 5 mM PVA-NO group had closed wounds whereas none in the 0.5 mMwere closed. By day 27, of four animals remaining in each group, one inthe 0.5 mM PVA-NO group, two in the control group and three in the 5 mMgroup had closed, epithelialized wounds. FIGS. 16A and 16B show thewound area and perimeter over time. Representative digital images of thewounds at days 10, 17, and 27 were also taken. Images of the wounds werecaptured every 2–3 days to quantify wound area and perimeter throughimage processing software. Day 27 was the endpoint of the wound healingstudy, with animals having closed, epithelialized wounds. Wound area andperimeter were similar in test and control groups.

Granulation tissue, characterized by proliferating fibroblasts and newlyformed microvasculature, was present in the open wound at days 8 and 15in all groups. FIG. 17 shows a graph of the granulation tissue thicknesscomparing the test and control groups. The results reflect the meanthickness and standard deviation of 3 measurements taken on each of twoserial sections (total of 6 measurements) within the region of the openwound. The three measurements were at a central point and 0.5 mm oneither side of this point. Granulation tissue tended to be thicker withincreasing NO concentration, however this difference was notstatistically significant. Representative histological sections ofgranulation tissue formation at days 8 and 15 in the control group andthe 5 mM PVA-NO group. Granulation tissue thickness was assessed throughanalysis of histological sections stained with hematoxylin and eosin.Representative sections were taken for treatment with a) controlhydrogels and b) 5 mM PVA-NO hydrogels at day 8, as well as treatmentwith c) control hydrogels and d) 5 mM PVA-NO hydrogels at day 15.Increased granulation tissue thickness was observed in wounds treatedwith NO-releasing hydrogels compared to controls.

Collagen deposition was assessed in histological sections stained withMasson's Trichrome at day 29, after wounds were completely closed andepithelialized. This was measured in a similar way to granulation tissueat a central point and 2 points on either side 0.5 mm of the centralpoint. Measurements were conducted on 4 sections from each animal. FIG.18 shows a graph of the collagen tissue thickness (mean±SD) in thecontrol and 5 mM test group. The collagen was significantly thicker inthe NO treated group (p<0.001).

Modifications and variations of the methods and materials describedherein will be obvious to those skilled in the art from the foregoingdetailed description and accompanying figures. These methods andmaterials are intended to be encompassed by the following claims.

1. A biocompatible, polymerizable, macromer composition comprising amacromer having at least one NO carrying region or NO modulatingcompound, wherein the NO or NO modulating compound is released from themacromer, and wherein the macromer further comprises one or more regionsselected from the group consisting of a water soluble region, a celladhesion ligand, and a polymerizable region.
 2. The macromer compositionof claim 1 wherein the macromer further comprises at least onedegradable region.
 3. The macromer composition of claim 1 wherein themacromer is water soluble.
 4. The macromer composition of claim 1wherein the macromer adheres to tissue.
 5. The macromer composition ofclaim 1 further comprising therapeutic, prophylactic or diagnosticagents selected from the group consisting of proteins, carbohydrates,nucleic acids, organic molecules, inorganic molecules, active cells,tissues and tissue aggregates.
 6. The macromer composition of claim 1wherein the macromer comprises a water soluble region, an NO carryingregion, a cell adhesion ligand, and a free radical polymerizable region.7. The macromer composition of claim 6 further comprising a degradableregion.
 8. A method for modulating NO levels in tissue comprisingadministering to the tissue any of the macromer compositions of any oneof claims 1–7.
 9. The macromer composition of claim 1 wherein the celladhesion ligand is RGD, RGDS or REDV.
 10. The macromer composition ofclaim 1 wherein the water soluble region is polyvinyl alcohol and thepolymerizable group is an acrylamide.
 11. The macromer composition ofclaim 1 wherein the macromer is an acryloyl-PEG-Cys-NO macromer.
 12. Themacromer composition of claim 1 wherein the macromer is anacryloyl-PEG-Lys.sub.5-NO macromer.
 13. The macromer composition ofclaim 1 wherein the macromer is a PEG-DETA-NO macromer.
 14. The macromercomposition of claim 1 wherein the macromer is a PVA-NH.sub.2-NOmacromer.
 15. The macromer composition of claim 1 wherein the macromeris a PVA-Cys-NO macromer.
 16. The macromer composition of claim 1wherein the macromer is a PVA-NO-bFGF macromer.
 17. The macromercomposition of claim 1 wherein the cell adhesion ligand is part of theNO-releasing macromer.
 18. The macromer composition of claim 1 whereinthe macromer composition is a blend of two or more macromers, wherein atleast one of the macromers has an NO carrying region or NO modulatingcompound, wherein the NO or NO modulating compound is released from themacromer composition, and wherein at least one of the macromers has atleast one cell adhesion ligand.
 19. The macrotner composition of claim1, further comprising one or more biologically active agents selectedfrom the group consisting of antibiotics, antiviral drugs, antifungaldrugs, anti-inflanunatory agents, hormones, growth factors, cytolcines,neuroactive agents, vasoconstrictors, molecules involved incardiovascular response, enzymes, antineoplastic agents, localanesthetics, antiangiogenic agents, antibodies, drugs affectingreproductive organs, and antisense oligonucleotides.
 20. The macromnercomposition of claim 1, further comprising diagnostic agents, whereinthe diagnostic agents are radioactive, bound to a chromogenic substrate,cleave the chroniogenic substrate, or detectable by imaging means. 21.The macromer composition of claim 20, wherein the imaging means areultrasound, x-ray, or MRI imaging means.